Two levels of cooperativeness in the binding of TodT to the tod operon promoter.
The TodS/TodT two-component system controls the expression of tod genes for toluene degradation in Pseudomonas putida. TodT binds to two pseudopalindromes at -106 (Box-1) and -85 (Box-2), as well as to a half-palindrome (Box-3), with respect to the main transcription initiation site in the PtodX promoter. TodT recognizes each half-palindrome in Boxes-1 and -2, but affinities for these sequences are lower than those for the pseudopalindromes, pointing towards positive cooperativeness in intrabox recognition. TodT's affinity for DNA fragments containing two vicinal boxes (either Boxes-1 and -2 or Boxes-2 and -3) is higher than its affinity for individual boxes, suggesting interbox cooperativeness. Similar patterns of cooperativeness were observed for the recombinant TodT DNA-binding domain [C-terminal TodT fragment (aa 154-206) (C-TodT)], suggesting important cooperativeness determinants in this domain. Occupation of PtodX by TodT is initiated at Box-1, and optimization of its palindromic order increases affinity in vitro; however, this does not result in enhanced in vivo gene expression. Mutations at either half of the Box-1 palindrome have no significant effects on transcriptional activity, whereas mutations in the entire Box-1 cause a 12-fold reduction. Using atomic force microscopy, we show that TodT induces a DNA hairpin bend at PtodX between Boxes-2 and -3, as supported by footprint studies showing a hyperreactive nucleotide at G -68. The N-terminal part of TodT seems to play a central role in hairpin formation, since C-TodT neither induces a bend nor causes G -68 hyperreactivity in footprints. This hairpin seems important for transcriptional activation, since C-TodT binding to PtodX does not stimulate transcription.
Lacal
Jesús
J
Department of Environmental Protection, Estación Experimental del Zaidín,Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain.
Guazzaroni
María-Eugenia
ME
Gutiérrez-del-Arroyo
Paloma
P
Busch
Andreas
A
Vélez
Marisela
M
Krell
Tino
T
Ramos
Juan L
JL
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D055029:Inverted Repeat Sequences / Q000235:genetics; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D009876:Operon / Q000235:genetics*; D010446:Peptide Fragments / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D016958:Pseudomonas putida / Q000235:genetics*; D013816:Thermodynamics; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010446:Peptide Fragments; C104650:TodT protein, Pseudomonas putida; D015534:Trans-Activators; D003850:Deoxyribonuclease I